Genes controlling plant cell wall formation

ABSTRACT

A Eucalyptus EST database was prepared and used for extracting genes specifically expressed in Eucalyptus reaction wood tissues using microarray analysis. As a result, genes were broadly classified into a gene cluster with a predominantly high expression, a gene cluster with a lower expression, and a gene cluster that is virtually unchanged, in Eucalyptus reaction wood as compared to ordinary trunks. It is thought that the gene cluster with predominantly high expression and the gene cluster with low expression can be used to control cell wall biosynthesis and wood fiber cell morphogenesis

TECHNICAL FIELD

The present invention relates to genes that control plant cell wallbiosynthesis and wood fiber cell morphogenesis, and their use.

BACKGROUND ART

The amount of wood consumed throughout the world continues to increaseeach year. In terms of use, consumption of wood for fuel accounts formore than half, and this amount is on the rise in developing regionseven at present. Although the amount of industrial materials, such aswood chips for lumber and paper production, consumed in developedregions has started to decrease slightly, the amounts consumed indeveloping countries and regions are increasing. Even though forests indeveloped regions have been considerably depleted due to conversion intoagricultural land and use as building materials, they have recentlystarted to increase slightly due to tree planting activities. Indeveloping regions, however, due to commercial logging by developedcountries in the past, and the increasing demand for domestic fuel andagricultural land in recent years accompanying population growth, arapid decrease in forestland area is continuing on a global scale. As aresult, problems such as global warming are occurring due to decreasedability to fix carbon dioxide. In recent years, afforestation projectsare being actively conducted throughout the world in an attempt toprovide a stable wood chip supply for the lumber and paper industries,while resolving these issues.

Human beings have used forest resources (wood biomass) in a diverserange of industrial fields such as papermaking, construction, animalfeed, and fuel for many years. Industries that use wood biomass arebeing recognized anew from the viewpoint of improving globalenvironmental issues, as resources that can be sustainably used in thefuture as well. Much hope is placed on these as circulatory-typeindustries based on the use of carbon sources as an alternative tocurrent fossil resources. As a specific example, Japanese papermanufacturers are actively promoting afforestation projects focusing onrapidly-growing tropical trees such as Eucalyptus and acacia, in orderto achieve a stable and continuous supply of wood biomass, the rawmaterial. As an example of the scale of these afforestation projects,Oji Paper Co., Ltd. is conducting afforestation over a wide area,focusing on the Pan-Pacific region such as Oceania and SoutheastAsia,aiming at 200,000 hectares of afforested land by 2010. This clearlydemonstrates that through large-scale afforestation at the commerciallevel, the paper industry is taking the initiative ahead of otherindustrial fields in recycled material production (biomass recycling)through industrial utilization and regeneration of biomass.

Along with the progress of these afforestation projects, if the amountand quality of woody cell wall components (cellulose, hemicellulose, andlignin) as well as fiber morphology (elongation of wood fiber cells)could be freely altered by artificially controlling the production ofwood biomass in trees, both quantitative increases and qualitativeimprovements in wood biomass can be expected. This in turn is hoped toexpand applications in energy usage and utilization as industrial rawmaterials in the future, thereby leading to replacement of currentfossil materials in various fields.

Cell walls and cellulose, the main component of cell walls, play animportant role in maintaining plant morphology. However, despite aconsiderable amount of time spent throughout the world on research toelucidate the mechanism of cellulose biosynthesis, as well genomic levelanalytical research on trees (Non-Patent Document 1), the details ofthis mechanism remain unclear. Recently, a glycosyl transferase gene,which is thought to catalyze the bonding of β-1,4 glucans serving as thebasic backbone of cellulose, was reported in cotton and Arabidopsisthaliana (see, for example, Non-Patent Document 2). In addition, as aresult of analyzing genes characteristic of wood formation, particularlythe secondary wall synthesis of the cell wall, using microarrays forpoplar vascular bundle tissues in different development stages, a knowncellulose synthetase was found (Non-Patent Document 3). However,significant progress is yet to be seen in research relating toregulation of the entire cellulose biosynthesis mechanism, and the like(Patent Document 1).

[Patent Document] Japanese Patent Kohyo Publication No. (JP-A)2002-510961 (unexamined Japanese national phase publicationcorresponding to a non-Japanese international publication)

[Non-patent Document 1] Genome Biol. 2002; 3(12):REVIEWS 1033.

[Non-patent Document 2] Burn et al. Plant Physiol. 129, 797-807, 2002.

[Non-patent Document 3] Hertzberg M, Aspeborg H, Schrader J, AnderssonA, Erlandsson R, Blomqvist K, Bhalerao R, Uhlen M, Teeri T T, LundebergJ, Sundberg B, Nilsson P, Sandberg G. A transcriptional roadmap to woodformation. Proc Natl Acad Sci USA. 2001 Dec. 4; 98(25):14732-7. Epub2001 Nov. 27.

[Non-patent Document 4] Szyjanowicz P M, McKinnon I, Taylor N C,Gardiner J, Jarvis M C, Turner S R. The irregular xylem 2 mutant is anallele of korrigan that affects the secondary cell wall of Arabidopsisthaliana. Plant J. 2004 March; 37(5):730-40.

[Non-patent Document 5] Nakazono M, Qiu F; Borsuk L A, Schnable P S.Laser-capture microdissection, a tool for the global analysis of geneexpression in specific plant cell types: identification of genesexpressed differentially in epidermal cells or vascular tissues ofmaize. Plant Cell. 2003 March; 15(3):583-96. Erratum in: Plant Cell.2003 April; 15(4):1049.

[Non-patent Document 6] Israelsson M, Eriksson M E, Hertzberg M,Aspeborg H, Nilsson P, Moritz T. Changes in gene expression in thewood-forming tissue of transgenic hybrid aspen with increased secondarygrowth. Plant Mol. Biol. 2003 July; 52(4):893-903.

[Non-patent Document 7] Gardiner J C, Taylor N G, Turner S R. Control ofcellulose synthase complex localization in developing xylem. Plant Cell.2003 August; 15(8):1740-8.

[Non-patent Document 8] Moller R, McDonald A G, Walter C, Harris P J.Cell differentiation, secondary cell-wall formation and transformationof callus tissue of Pinus radiata D. Don. Planta. 2003 September;217(5):736-47. Epub 2003 Jun. 13.

[Non-patent Document 9] Joshi C P. Xylem-specific and tensionstress-responsive expression of cellulose synthase genes from aspentrees. Appl Biochem Biotechnol. 2003 Spring; 105-108:17-25.

[Non-patent Document 10] Li L, Zhou Y, Cheng X, Sun J, Marita J M, RalphJ, Chiang V L. Combinatorial modification of multiple lignin traits intrees through multigene cotransformation. Proc Natl Acad Sci USA. 2003Apr. 15; 100(8):4939-44. Epub 2003 Mar. 31.

[Non-patent Document 11] Lorenz W W, Dean J F. SAGE profiling anddemonstration of differential gene expression along the axialdevelopmental gradient of lignifying xylem in loblolly pine (Pinustaeda). Tree Physiol. 2002 April; 22(5):301-10.

[Non-patent Document 12] Demura T, Tashiro G, Horiguchi G, Kishimoto N,Kubo M, Matsuoka N, Minami A, Nagata-Hiwatashi M, Nakamura K, Okamura Y,Sassa N, Suzuki S, Yazaki J, Kikuchi S, Fukuda H. Visualization bycomprehensive microarray analysis of gene expression programs duringtransdifferentiation of mesophyll cells into xylem cells. Proc Natl AcadSci USA. 2002 Nov. 26; 99(24):15794-9. Epub 2002 Nov. 18.

[Non-patent Document 13] Aharoni A, Keizer L C, Van Den Broeck H C,Blanco-Portales R, Munoz-Blanco J, Bois G; Smit P, De Vos R C, O'ConnellA P. Novel insight into vascular, stress, and auxin-dependentand-independent gene expression programs in strawberry, anon-climacteric fruit. Plant Physiol. 2002 July; 129(3):1019-31.

DISCLOSURE OF THE INVENTION

Japan lacks natural resources and is dependent on fossil resources suchas petroleum and natural gas even now. In order to change thesecircumstances using new technology, the recycling of trees (woodbiomass) is currently considered to be instrumental. Although foreigncountries differed in their approach towards forestry in the past, theestablishment of technologies relating to the effective use of woodbiomass was placed as an important topic at the beginning of the currentcentury. In fact, several countries have begun research on target woodspecies (pine trees of needle-leaved trees and poplar of broad-leavedtrees in the U.S., spruce and poplar in Canada, and poplar inScandinavia) using genomic analyses as national projects. It is wellknown that the U.S. and Europe are currently ahead of research on basictechnologies related to gene recombination of important crop varietiesinvolved in food production. Learning from this, there is an extremelyhigh need to identify genes that control plant cell wall componentbiosynthesis and wood fiber cell morphogenesis, so that Japan can becomethe technological powerhouse it was before, or to at least keep up withforeign countries, and be involved in the production of recycledmaterial through the utilization of wood biomass on a global scale.

Considering the aforementioned circumstances, an objective of thepresent invention is to provide genes that control plant cell wallcomponent biosynthesis and wood fiber cell morphogenesis, plasmidscomprising these genes, and plant cells, microorganisms, or plantstransformed by the plasmids.

As a result of extensive research to achieve the aforementionedobjective, the present inventor concluded that the acquisition of a genecluster that controls wood biomass formation and comprehensive analysesrelating to its expression and function should be carried out based on agenomic approach. Namely, it was concluded that it is desirable to use amethod of systematically acquiring and analyzing a target gene clusterat a time when formation of a specific tissue (particularly the cellwall) is active and cellulose is specifically biosynthesized. Moreover,it was concluded that, in order to provide a plant with characteristicsuseful for human use by artificially controlling the expression of analtered gene using genetic engineering technology, it is necessary toidentify a gene cluster specific to various tissues that selectivelyexpress the novel characteristics in suitable plant tissues.

In the implementation of this research, resources for analyses wereprepared using Eucalyptus. More specifically, various gene libraries andan EST database were prepared for each of trunk, leaf, and root tissues.Gene libraries and mutants involved in cell wall biosynthesis werealready present for Arabidopsis thaliana, a plant that is widely used asa plant model throughout the world. These causative genes have alreadybeen analyzed.

The present inventor extracted genes specifically expressed inEucalyptus reaction wood forming tissue by microarray analysis, usingthe aforementioned Eucalyptus EST database. As a result, the genes werebroadly classified into a gene cluster demonstrating predominantly highexpression, a gene cluster demonstrating low expression, and a genecluster which demonstrated virtually no changes in expression, inEucalyptus reaction wood as compared with ordinary trunk wood. The genecluster that demonstrated predominantly high expression and the genecluster that demonstrated low expression may be used to control cellwall component biosynthesis and wood fiber cell morphgenesis. Inparticular, the gene cluster that demonstrated predominantly highexpression, may be involved in cell wall component biosynthesis and woodfiber cell morphogenesis, and may be used to promote cell wall componentbiosynthesis and wood fiber cell morphogenesis.

One of the outcomes of the gene clusters that control cell wallcomponent biosynthesis and wood fiber cell morphogenesis obtained by thepresent invention, as well as techniques for their overall control,would be various quantitative and qualitative changes (such as highcellulose content, low lignin content, thick or thin cell walls, andlong or short fiber lengths) in the characteristics of novel transgenicEucalyptus varieties obtained by using these gene clusters.

Namely, the present invention relates to genes that control plant cellwall biosynthesis and wood fiber cell motphogenesis, and provides thefollowing [1] to [14].

[1] A DNA whose expression increases during plant cell wall biosynthesisand wood fiber cell morphogenesis, wherein the DNA is described in (a)or (b) below:

-   -   (a) a DNA that hybridizes under stringent conditions with a DNA        comprising a nucleotide sequence described in any one of SEQ ID        NOs: 1 to 862; or,    -   (b) a DNA encoding a protein having 50% or more homology with a        protein comprising an amino acid sequence encoded by the DNA of        (a).        [2] The DNA of [1], wherein expression increases in plant        reaction wood forming tissue.        [3] A DNA whose expression decreases during plant cell wall        biosynthesis and wood fiber cell morphogenesis, wherein the DNA        is described in (a) or (b) below:    -   (a) a DNA that hybridizes under stringent conditions with a DNA        comprising a nucleotide sequence described in any one of SEQ ID        NOs: 863 to 1731; or,    -   (b) a DNA encoding a protein having 50% or more homology with a        protein comprising an amino acid sequence encoded by the DNA of        (a).        [4] The DNA of [3], wherein expression decreases in plant        reaction wood forming tissue.        [5] The DNA of any one of [1] to [4], wherein the plant is        Eucalyptus.        [6] A DNA encoding a protein comprising an amino acid sequence        in which one or more nucleotides are substituted, deleted, added        and/or inserted in an amino acid sequence encoded by the DNA of        any one of [1]to [5].        [7] A promoter DNA of the DNA of any one of [1] to [5].        [8] A DNA described in any one of (a) to (e) below:    -   (a) a DNA encoding an antisense RNA complementary to a        transcription product of the DNA of any one of [1] to [5];    -   (b) a DNA encoding an RNA having ribozyme activity that        specifically cleaves a transcription product of the DNA of any        one of [1] to [5];    -   (c) a DNA encoding an RNA that suppresses expression of the DNA        of any one of [1] to [5] by RNAi effects;    -   (d) a DNA encoding an RNA that suppresses expression of the DNA        of any one of [1] to [5] by co-suppression effects; and,    -   (e) a DNA encoding a protein having a dominant negative trait        against a transcription product of the DNA of any one of claims        [1] to [5].        [9] A recombinant vector comprising the DNA of any one of [1] to        [6] or [8].        [10] A microorganism retaining a plasmid comprising the promoter        DNA of [7] or the vector of [9].        [11] A transgenic plant cell introduced with the vector of [9].        [12] A transgenic plant that is re-differentiated from the        transgenic plant cell of [11].        [13] A transgenic plant that is a progeny or a clone of the        transgenic plant of [12].        [14] A breeding material of the transgenic plant of [12] or        [13].

The present inventor discovered DNA with varied expression in Eucalyptusreaction wood forming tissue. Cell wall component biosynthesis and woodfiber cell morphogenesis are known to take place in plant reaction woodforming tissue (although general descriptions on reaction wood canalways be found in technical literature relating to wood, a paper byBaba, et al. (Mokuzai Gakkaishi 42, 795-798, 1996) describes detaileddata on the chemical and structural properties of reaction wood inEucalyptus). Based on the aforementioned findings, the present inventionprovides DNA whose expression varies during plant cell wall componentbiosynthesis and wood fiber cell morphogenesis.

A DNA whose expression varies during plant cell wall componentbiosynthesis and wood fiber cell morphogenesis of the present inventionmay be used to control plant cell wall component biosynthesis and woodfiber cell morphogenesis. In addition, a DNA whose expression increasesduring plant cell wall component biosynthesis and wood fiber cellmorphogenesis is particularly thought to be involved in plant cell wallcomponent biosynthesis and wood fiber cell morphogenesis, and thereforemay be used to promote cell wall component biosynthesis and wood fibercell morphogenesis. On the other hand, a DNA whose expression decreasesduring plant cell wall component biosynthesis and wood fiber cellmorphogenesis may be involved in the control of tissue-specific ortime-specific expression, by basically suppressing genes involved incell wall component biosynthesis and wood fiber cell morphogenesisthrough some sort of a mechanism. Thus, these DNAs may be used toenhance cell wall component biosynthesis and wood fiber cellmorphogenesis by artificially prompting their decrease.

Controlling cell wall component biosynthesis and wood fiber cellmorphogenesis in plants has various important significances inindustrial and agricultural fields. For example, alteration of plantcell wall components is significant in terms of economical efficiencyand profitability, by enhancing the supply of high-quality fiber rawmaterials such as pulp as a result of increasing cellulose andhemicellulose contents, and by improving the digestion and absorptionefficiencies of useful agricultural crops and feed products. Inaddition, changing the structure of polysaccharides, which is a cellwall component, may lead to the production of raw material plants havingnew industrial values. Moreover, alteration of cell morphology issignificant in terms of, for example, improving the fibercharacteristics of fiber raw materials such as pulp.

In addition, a DNA of the present invention whose expression variesduring plant cell wall component biosynthesis and wood fiber cellmorphogenesis can also be used as a specific marker for identifyingcells and tissues in which cell wall component biosynthesis and woodfiber cell morphogenesis are taking place.

There are no particular limitations on the plants from which the DNA ofthe present invention is derived from. Examples include usefulagricultural crops such as grains, vegetables, and fruits (includingfeed crops), fiber raw material plants such as pulp, and plants valuedfor their aesthetic beauty such as foliage plants. There are noparticular limitations on such plants, and examples include Eucalyptus,pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat,barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean,sunflower, cotton, orange, grape, peach, pear, apple, tomato, Chinesecabbage, cabbage, radish, carrot, squash, cucumber, melon, parsley,orchid, chrysanthemum, lily, and saffron.

An example of a DNA of the present invention is a DNA that hybridizesunder stringent conditions with a DNA comprising a nucleotide sequencedescribed in any one of SEQ ID NOs: 1 to 1731. Among these, a DNA thathybridizes under stringent conditions with a DNA comprising a nucleotidesequence described in any one of SEQ ID NOs: 1 to 862 is a DNA whoseexpression increases during plant cell wall component biosynthesis andwood fiber cell morphogenesis. In addition, a DNA that hybridizes understringent conditions with a DNA comprising a nucleotide sequencedescribed in any one of SEQ ID NOs: 863 to 1731 is a DNA whoseexpression decreases during plant cell wall component biosynthesis andwood fiber cell morphogenesis.

Stringent hybridization conditions comprise allowing to stand overnightat 60° C. in 0.1×SSC solution, or conditions yielding stringenciessimilar to these. Under these conditions, a DNA that hybridizes with aDNA comprising a nucleotide sequence described in any one of SEQ ID NOs:1 to 1731 can be isolated.

More specifically, a continuous proximal sequence can be easily acquiredby extracting a DNA from a plant, constructing a gene library, andscreening under similar conditions, or by carrying out the TAIL-PCRmethod established by Ryu, et al. on the extracted DNA using anarbitrary sequence of about 20 mer from a 60 mer sequence (nucleotidesequence described in any one of SEQ ID NOs: 1 to 1731) for the primer.

In addition, the present invention provides a DNA that encodes a proteinhaving 50% or more homology with a protein comprising an amino acidsequence encoded by a DNA that hybridizes under stringent conditionswith a DNA comprising a nucleotide sequence described in any one of SEQID NOs: 1 to 1731. Such DNA can be isolated by methods commonly known topersons skilled in the art. Examples include, methods that usehybridization technology (Southern, E M., J Mol Biol, 1975, 98, 503) orpolymerase chain reaction (PCR) technology (Saiki, R K et al., Science,1985, 230, 1350., Saiki, RK. et al., Science, 1988, 239, 487). Namely,isolation of a DNA having high homology with a DNA that hybridizes understringent conditions with a DNA comprising a nucleotide sequencedescribed in any one of SEQ ID NOs: 1 to 1731 from a plant by using aDNA or a portion thereof that hybridizes under stringent conditions witha DNA comprising a nucleotide sequence described in any one of SEQ IDNOs: 1 to 1731 as a probe, or by using an oligonucleotide thatspecifically hybridizes with a DNA that hybridizes under stringentconditions with a DNA comprising a nucleotide sequence described in anyone of SEQ ID NOs: 1 to 1731 as a primer, are tasks that can beroutinely carried out by one skilled in the art.

Hybridization reactions to isolate such DNAs are preferably conductedunder stringent conditions. Stringent hybridization conditions of thepresent invention include conditions such as 6 M urea, 0.4% SDS, and0.5×SSC, and those conditions yielding similar stringencies to these.DNAs with higher homology are expected to be isolated when hybridizationis performed under more stringent conditions, for example, 6 M urea,0.4% SDS, and 0.1×SSC. DNAs thus isolated are thought to have highhomology, at an amino acid level, with amino acid sequences encoded byDNAs that hybridize under stringent conditions to DNAs comprising anyone of the nucleotide sequences described in SEQ ID NOs: 1 to 1731.Herein, high homology means an identity over the entire amino acidsequence of at least 50% or above, more preferably 70% or above, evenmore preferably 80% or above, still more preferably 90% or above, evenstill more preferably 95% or above, and most preferably 98% or above.Such DNAs comprise degenerative variants of the DNAs that hybridizeunder stringent conditions with the DNAs comprising any one of thenucleotide sequences described in SEQ ID NOs: 1 to 1731.

The degree of homology of one amino acid sequence or nucleotide sequenceto another can be determined using the BLAST algorithm by Karlin andAltschul (Proc. Natl. Acad. Sci. USA, 1990, 87, 2264-2268., Karlin, S. &Altschul, S F., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873). Programssuch as BLASTN and BLASTX, developed based on the BLAST algorithm(Altschul, SF. et al., J. Mol. Biol., 1990, 215, 403.), are also used.To analyze a nucleotide sequence according to BLASTN, parameters areset, for example, at score=100 and word length=12. On the other hand,parameters used for the analysis of amino acid sequences by BLASTX are,for example, score=50 and word length=3. The default parameters for eachprogram are used when using the BLAST and Gapped BLAST programs.Specific techniques of such analyses are known in the art (seehttp://www.ncbi.nlm.nih.gov.)

In addition, the DNA of the present invention comprises a DNA thatencodes a protein comprising an amino acid sequence encoded by a DNAthat hybridizes under stringent conditions with a DNA comprising anucleotide sequence described in any one of SEQ ID NOs: 1 to 1731, or anamino acid sequence in which one or more amino acids are substituted,deleted, added and/or inserted in an amino acid sequence having 50% ormore homology with a protein comprising the amino acid sequence.

An example method widely known to persons skilled in the art forpreparing the aforementioned DNA is a method in which a mutation isintroduced into a DNA by site-directed mutagenesis (Kramer, W. & Fritz,H J., Methods Enzymol, 1987, 154, 350).

Modification of amino acids in proteins is usually in the range of notmore than 50 in the whole number of amino acids, preferably not morethan 30, more preferably not more than 10, and even more preferably, notmore than 3 amino acids. Amino acid modifications may be performed, forexample, in the case of mutations and substitutions, using a“Transformer Site-directed Mutagenesis Kit” or “ExSite PCR-BasedSite-directed Mutagenesis Kit” (Clontech), and, in the case ofdeletions, using a “Quantum leap Nested Deletion Kit” (Clontech) and thelike.

A nucleotide sequence may be mutated without causing mutations in theamino acids within a protein (degenerative mutation). The presentinvention also comprise such degenerative mutant DNAs.

There is no particular limitation on the type of DNAs of this inventionas long as they are capable of encoding the proteins of this invention,and include genomic DNA, cDNA, chemically synthesized DNA, etc. GenomicDNAs may be prepared by conducting PCR (Saiki et al., Science, 1988,239, 487) using as a template genomic DNA prepared according to a methoddescribed in literature (Rogers and Bendich, Plant Mol. Biol., 1985, 5,69) and primers prepared based on a nucleotide sequence of a DNA of thisinvention (e.g. a nucleotide sequence set forth in any one of SEQ IDNOs: 1 to 1731). Furthermore, cDNA may be prepared according to thestandard method (Maniatis et al., “Molecular Cloning”, Cold SpringHarbor Laboratory Press), by preparing mRNA from plants, performingreverse transcription, and conducting PCR using primers similar to thosedescribed above. Genomic DNA and cDNA may also be prepared byconstructing a genomic DNA library or a cDNA library according to thestandard method, and screening this library using a probe, for example,one synthesized based on the a nucleotide sequence of a DNA of thepresent invention (e.g. the sequence set forth in any one of SEQ ID NOs:1 to 1731). The DNA thus obtained may be easily sequenced using, forexample, the “Sequencer Model 373” (ABI).

In addition, the present invention provides a promoter DNA of a DNA ofthe present invention. Such promoter DNA include a promoter DNAadjoining a gene that is specifically expressed by a plant (particularlya tree) obtained according to the present invention, during cell wallformation and/or specifically expressed during cellulose biosynthesis.Here, “promoter DNA” refers to a DNA comprising a specific nucleotidesequence required to start mRNA synthesis (transcription) using DNA as atemplate, and comprises a DNA present in nature, as well as a DNAproduced by recombination or other artificial modification procedure.

A promoter of the present invention can be produced and used asdescribed below. A DNA is extracted and purified from the tissue of atarget Eucalyptus plant. Various methods can be used for the DNApreparation, including commercial kits such as the ISOPLANT Kit (NipponGene).

An oligonucleotide can then be produced from two arbitrary locationsbased on the nucleotide sequence of Eucalyptus cDNA that has alreadybeen successfully isolated by the present inventor using the resultingDNA as material. Genomic DNA corresponding to the selected EucalyptuscDNA can then be easily produced by PCR using this oligonucleotide asprimer. An upstream DNA of the gene can be isolated by PCR using anoligonucleotide primer produced based on the nucleotide sequence of thegene (Inverse-PCR and Anchor PCR/TAIL PCR (Shimamoto Ko, et al., ed.,“Shinpan Shokubutsuno PCR Jikken Protocol (PCR Experimental Protocolsfor Plants, New edition)” (Bessatu Shokubutsu Saibou Kogaku (Plant CellTechnology Supplementary Volume), Shokubutsu Saibou Kogaku (Plant CellTechnology) Series 7), Shujunsha Co., Ltd., July 1997)), or byhybridization using a DNA sequence of the gene as probe.

A genomic DNA library can also be used for the Eucalyptus DNA. A genomicDNA library is obtained by inserting a DNA extracted from Eucalyptusinto a cloning vector, such as various types of vectors derived fromλDNA, cosmid vector, or TAC vector (Liu, et al. (1999), Proc. Natl.Acad. Sci. USA, Vol. 96, p. 6535), and then transforming Escherichiacoli.

Hybridization techniques can be used to screen the genomic DNA library.A Eucalyptus cDNA sequence successfully isolated previously by thepresent inventor can be used for the probe. A clone comprising a DNAsequence homologous to the gene is isolated by screening theaforementioned DNA library using this probe. The structure of the clonedDNA is determined by producing a restriction enzyme cleavage map,determining nucleotide sequences and so forth, to specify the sequencepresent upstream from the gene. This upstream sequence preferablycontains a TATA box sequence, and is at least several hundred bp toseveral kbp in size. This sequence is then cut out by a suitablerestriction enzyme, and sub-cloned to other plasmid vectors and so forthas necessary.

The promoter activity of the aforementioned sequence can be analyzed asdescribed below. For example, a vector such as pBI101 comprising areporter gene is used, wherein the aforementioned sequence is subclonedsuch that it is linked upstream of the reporter gene. E. coliβ-glucuronidase (GUS) is used as the reporter gene in the pBI101 vector.Gene expression can be monitored at the tissue level by using5-bromo-4-chloro-3-β-D-glucuronic acid (X-gluc) as substrate, since agene product of indigotin is formed as a blue precipitate as a result ofsubstrate degradation. In addition, if4-methyl-umbelliferyl-β-D-glucuronide (4MUG) is used for the substrate,gene expression can be quantified according to the fluorescence producedby the gene product. Furthermore, chloramphenicol acetyl transferasegene, luciferase gene, green fluoroscein protein gene and so forth canalso be used for the reporter gene, in addition to the GUS gene.

A chimeric gene construct produced as described above can be introducedinto, for example, a plant such as Arabidopsis thaliana mediated by anAgrobacterium, to analyze its function. When using pBI101 for thevector, a recombinant plasmid comprising the chimeric gene is introducedinto, for example, Agrobacterium tumefaciens strain MP90 usingelectroporation, and the resulting transformant is infected into anArabidopsis thaliana plant by, for example, the floral dip method(Shimamoto Ko et al., ed., “Model Shokubutuno Jikken Protocol(Experimental Protocols for Model Plants)” (Bessatu Shokubutu SaibouKogaku (Plant Cell Technology Supplementary Volume), Shokubutu SaibouKogaku (Plant Cell Technology) Series 4), Shujunsha Co., Ltd., April,1996)). Seeds from the infected plant are seeded in medium containingagents such as kanamycin based on the vector used, to obtain atransgenic plant that has become drug-resistant as a result of geneintroduction. Expression of the GUS reporter gene is then analyzed usingthis transgenic plant. A promoter of the present invention or anexpression vector comprising the same, can be used as described below. Adesired gene downstream from a promoter of the present invention (e.g. achimeric gene linked to a gene involved in a certain type of response toosmotic pressure stress) is inserted into, for example, a pBI101 vectorto construct an expression vector. This vector is then introduced into,for example, a tobacco plant mediated by Agrobacterium. The resultingtransgenic plant is expected to be able to grow even under salt damageor in dry areas, as a result of gene expression in roots subjected to anenvironment with osmotic pressure stress, due to the action of thepromoter of the present invention. In this case, unlike the 35Spromoter, it is expected that other undesirable traits will not emerge,because gene expression in unwanted tissues will not occur.

Genes that can be controlled with a promoter of the present inventionare not limited to the aforementioned specific gene. In addition, thefunction of a promoter of the present invention can be altered bycoupling another expression regulating sequence to a promoter of thepresent invention. Examples of such expression regulating sequencesinclude enhancer sequences, repressor sequences, and insulatorsequences. A promoter of the present invention comprises severalcis-element sequences that control the expression of genes involved intrunk-specificity and cell wall biosynthesis as functionalcharacteristics. A portion of a promoter of the present invention can beinserted into and coupled with another promoter to alter the function ofthat promoter, with the aim of utilizing a cis-element sequencecomprised in a promoter of the present invention.

In addition, the present invention provides a DNA for suppressing theexpression of a DNA encoding a protein that controls plant cell wallcomponent biosynthesis and wood fiber cell morphogenesis. Preferredembodiments of DNA for suppressing the expression of an endogenous genecan be exemplified by a DNA that encodes an antisense RNA complementaryto a transcription product of a DNA of the present invention, a DNA thatencodes an RNA having ribozyme activity that specifically cleaves atranscription product of a DNA of the present invention, a DNA thatencodes an RNA that suppresses expression of a DNA of the presentinvention by RNAi effects or co-suppression effects, and a DNA thatencodes a protein having dominant native trait against a transcriptionproduct of a DNA of the present invention. The aforementioned“suppressing the expression of an endogenous gene” comprise suppressionof gene transcription and/or suppression of translation to a proteinencoded by the gene. In addition, it also comprises not only thecomplete cessation of gene expression, but also a decrease inexpression.

Antisense techniques are the most commonly used methods in the art tosuppress the expression of a specific endogenous gene in plants. Eckeret al. were the first to demonstrate the antisense effect of anantisense RNA introduced into plant cells by electroporation (Ecker, JR.& Davis, R W., Proc. Natl. Acad. Sci. USA, 1986, 83, 5372). Thereafter,it was reported that the expression of antisense RNAs reduced targetgene expression in tobacco and petunias (van der Krol A R. et al.,Nature, 1988, 333, 866.). Antisense techniques have now been establishedas a means for suppressing target gene expression in plants.

Multiple factors act in the suppression of target gene expression byantisense nucleic acids. These include: inhibition of transcriptioninitiation by triple strand formation; inhibition of transcription byhybrid formation at a site where the RNA polymerase has formed a localopen loop structure; transcription inhibition by hybrid formation withthe RNA being synthesized; inhibition of splicing by hybrid formation atan intron-exon junction; inhibition of splicing by hybrid formation at asite of spliceosome formation; inhibition of mRNA translocation from thenucleus to the cytoplasm by hybrid formation with mRNA; inhibition ofsplicing by hybrid formation at a capping site or poly A addition site;inhibition of translation initiation by hybrid formation at atranslation initiation factor binding site; inhibition of translation byhybrid formation at a ribosome binding site near the initiation codon;inhibition of peptide chain elongation by hybrid formation in atranslated region or at an mRNA polysome binding site; and inhibition ofgene expression by hybrid formation at a site of interaction betweennucleic acids and proteins. These antisense nucleic acids suppresstarget gene expression by inhibiting various processes such astranscription, splicing, or translation (Hirashima and Inoue, “ShinSeikagaku Jikken Koza (New Biochemistry Experimentation Lectures) 2,Kakusan (Nucleic Acids) IV, Idenshi No Fukusei To Hatsugen (Replicationand Expression of Genes),” Nihon Seikagakukai Hen (The JapaneseBiochemical Society), Tokyo Kagaku Dozin, pp. 319-347, (1993)).

The antisense sequences of the present invention can suppress targetgene expression by any of the above mechanisms. In one embodiment, anantisense sequence designed to be complementary to an untranslatedregion near the 5′ end of the mRNA of a gene is thought to effectivelyinhibit translation of that gene. Sequences complementary to codingregions or to an untranslated region on the 3′ side can also be used.Thus, the antisense DNAs used in the present invention include both DNAscomprising antisense sequences against untranslated and translatedregions of the gene. The antisense DNAs to be used are conjugateddownstream of an appropriate promoter, and are preferably conjugated tosequences containing the transcription termination signal on the 3′side. DNAs thus prepared can be transformed into a desired plant byknown methods. The sequences of the antisense DNAs are preferablysequences complementary to an endogenous gene of the plant to betransformed, or a part thereof, but need not be perfectly complementaryso long as they can effectively suppress the gene's expression. Thetranscribed RNAs are preferably at least 90%, and more preferably atleast 95% complementary to the transcribed product of the target gene.In order to effectively suppress the expression of a target gene bymeans of an antisense sequence, antisense DNAs should be at leastnucleotides long, more preferably at least 100 nucleotides long, andstill more preferably at least 500 nucleotides long. However, theantisense DNAs to be used are generally shorter than 5 kb, andpreferably shorter than 2.5 kb.

DNAs encoding ribozymes can also be used to suppress the expression ofendogenous genes. A ribozyme is an RNA molecule comprising catalyticactivity. There are many ribozymes comprising various activities, andamong them, research focusing on ribozymes as RNA-cleaving enzymes hasenabled the design of ribozymes that cleave RNAs site-specifically.While some ribozymes of the group I intron type or the M1 RNA containedin RNaseP consist of 400 nucleotides or more, others belonging to thehammerhead-type or the hairpin-type comprise an activity domain of about40 nucleotides (Makoto Koizumi and Eiko Ohtsuka, Tanpakushitsu KakusanKohso (Nucleic acid, Protein, and Enzyme), 1990, 35, 2191).

The self-cleavage domain of a hammerhead-type ribozyme cleaves at the 3′side of C15 of the G13U14C15 sequence, and formation of a nucleotidepair between U14 and A9 at the ninth position is considered to beimportant for this ribozyme activity. It has been shown that cleavagemay also occur when the 15th nucleotide is A15 or U15 instead of C15(Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). If a ribozyme isdesigned to comprise a substrate-binding site complementary to the RNAsequences adjacent to the target site, one can create arestriction-enzyme-like RNA-cleaving ribozyme which recognizes the UC,UU, or UA sequence within a target RNA (Koizumi M. et al., FEBS Lett,1988, 239, 285; Makoto Koizumi and Eiko Ohtsuka, Tanpakushitsu KakusanKohso (Nucleic acid, Protein, and Enzyme), 1990, 35, 2191; Koizumi M. etal., Nucleic Acids Res., 1989, 17, 7059). For example, in the codingregion of DNAs that encode proteins that control plant cell wallcomponent biosynthesis and wood fiber cell morphogenesis, there are anumber of sites that can be used as targets.

Hairpin-type ribozymes are also useful in the present invention. Theseribozymes can be found, for example, in the minus strand of satelliteRNA in tobacco ringspot virus (Buzayan J M., Nature, 1986, 323, 349).Ribozymes that cleave RNAs target-specifically have also been shown tobe produced from hairpin-type ribozymes (Kikuchi Y & Sasaki N., NucleicAcids Res, 1991, 19, 6751; Yo Kikuchi, Kagaku To Seibutsu (Chemistry andBiology), 1992, 30, 112.).

Transcription is enabled in plant cells by fusing a ribozyme, designedto cleave a target, with a promoter such as the cauliflower mosaic virus35S promoter, and with a transcription termination sequence. If extrasequences have been added to the 5′ end or the 3′ end of the transcribedRNA, ribozyme activity can be lost. In such cases, one can place anadditional trimming ribozyme, which functions in cis, on the 5′ or the3′ side of the ribozyme portion, in order to precisely cut the ribozymeportion from the transcribed RNA containing the ribozyme (Taira, K. etal., Protein Eng, 1990, 3, 733., Dzianott, A M. & Bujarski, J J., ProcNatl Acad Sci USA, 1989, 86, 4823., Grosshans, C A. & Cech, T R., NuclAcids Res, 1991, 19, 3875., Taira, K. et al., Nucl Acids Res, 1991, 19,5125.). Even greater effects can be achieved by arranging thesestructural units in tandem, enabling multiple sites within a target geneto be cleaved (Yuyama, N. et al., Biochem Biophys Res Commun, 1992, 186,1271.). Thus, using these ribozymes, the transcription products of atarget gene of the present invention can be specifically cleaved,thereby suppressing expression of the gene.

Endogenous gene expression can also be suppressed by RNA interference(RNAi), using double-stranded RNAs that comprise a sequence identical orsimilar to a target gene. RNAi refers to the phenomenon in which adouble-stranded RNA comprising a sequence identical or similar to atarget gene sequence is introduced into cells, thereby suppressingexpression of both the exogenous gene introduced and the targetendogenous gene. The details of the RNAi mechanism are unclear, but itis thought that an introduced double-stranded RNA is first degraded intosmall pieces, which somehow serve as a target gene indicator, resultingin degradation of the target gene. RNAi is known to be effective inplants as well (Chuang, CF. & Meyerowitz, E M., Proc Natl Acad Sci USA,2000, 97, 4985.). For example, in order to use RNAi to suppress theexpression of DNAs encoding the proteins that control plant cell wallcomponent biosynthesis and wood fiber cell morphogenesis in plants,nucleotide sequences described in any one of SEQ ID NOs: 1 to 1731, ordouble-stranded RNAs comprising a sequence similar to these DNAs, can beintroduced into the plants in question. Genes used for RNAi need not becompletely identical to a target gene; however, they should comprisesequence identity of at least 70% or above, preferably 80% or above,more preferably 90% or above, and most preferably 95% or above. Sequenceidentity can be determined by an above-described method.

Suppression of endogenous gene expression can be achieved byco-suppression, through transformation with a DNA comprising a sequenceidentical or similar to a target gene sequence. “Co-suppression” refersto the phenomenon wherein transformation is used to introduce plantswith a gene comprising a sequence identical or similar to a targetendogenous gene sequence, thereby suppressing expression of both theexogenous gene introduced and the target endogenous gene. Although thedetails of the co-suppression mechanism are unclear, at least a part isthought to overlap with the RNAi mechanism. Co-suppression is alsoobserved in plants (Smyth D R., Curr. Biol., 1997, 7, R793.,Martienssen, R., Curr. Biol., 1996, 6, 810). For example, if one wishesto obtain a plant in which a DNA encoding proteins that control plantcell wall component biosynthesis and wood fiber cell morphogenesis isco-suppressed, the plant in question can be transformed with a vectorDNA designed to express the DNA encoding the protein, or a DNAcomprising a similar sequence. Genes for use in co-suppression do notneed to be completely identical to a target gene, but should comprisesequence identity of at least 70% or above, preferably 80% or above,more preferably 90% or above, and most preferably 95% or above. Sequenceidentity may be determined by an above-described method.

Moreover, suppression of the expression of an endogenous gene in thepresent invention can also be achieved by transforming a plant with agene that encodes a protein having a dominant native trait against aprotein that encodes a target gene. A “gene that encodes a proteinhaving a dominant native trait” refers to a gene having a function thateliminates or decreases the activity of an endogenous wild type proteininherently possessed by a plant, by causing expression of the gene.

In addition, the present invention provides recombinant vectorscomprising the aforementioned DNA. There are no particular limitationson the vectors of the present invention provided they comprise apromoter sequence that is transcribable in plant cells and a terminatorsequence comprising a polyadenylation site required for stabilizing thetranscription product. Examples include vectors that can be amplified inE. coli such as a pUC derivative, and shuttle vectors such as pBI101(Clontech) that can be amplified in both E. coli and Agrobacterium. Inaddition, plant viruses such as the cauliflower mosaic virus can also beused as a vector.

A vector of the present invention can be obtained by coupling orinserting a promoter DNA, for constant or inductive expression of apromoter DNA of the present invention or a desired gene at apredetermined site of a vector. Furthermore, the promoter is insertedinto the vector according to methods normally used for inserting genesinto vectors. An expression vector for gene expression can be obtainedby functionally connecting a desired gene to a promoter of thisrecombinant vector.

Promoters for constant expression are exemplified by the 35S promoter ofcauliflower mosaic virus (Odell et al., Nature, 1985, 313, 810), theactin promoter of rice (Zhang et al., Plant Cell, 1991, 3, 1155), theubiquitin promoter of corn (Cornejo et al., Plant Mol. Biol., 1993, 23,567), etc. Furthermore, promoters for inductive expression areexemplified by promoters that are expressed by extrinsic factors such asinfection and invasion of filamentous fungi, bacteria, and viruses, lowtemperature, high temperature, drought, ultraviolet irradiation,spraying of particular compounds, and the like. Such promoters areexemplified by the chitinase gene promoter of rice (Xu et al., PlantMol. Biol., 1996, 30, 387.) and tobacco PR protein gene promoter(Ohshima et al., Plant Cell, 1990, 2, 95.) expressed by the infectionand invasion of filamentous fungi, bacteria and viruses, the “lip 19”gene promoter of rice induced by low temperature (Aguan et al., Mol. GenGenet., 1993, 240, 1.), “hsp 80” and “hsp 72” gene promotors of riceinduced by high temperature (Van Breusegem et al., Planta, 1994, 193,57.), “rab 16” gene promoter of Arabidopsis thaliana induced by dryness(Nundy et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 1406), chalconesynthase gene promoter of parsley induced by ultraviolet irradiation(Schulze-Lefert et al., EMBO J., 1989, 8, 651.), alcohol dehydrogenasegene promoter of corn induced by anaerobic conditions (Walker et al.,Proc. Natl. Acad. Sci. USA, 1987, 84, 6624) and so on. In addition, thechitinase gene promoter of rice and PR protein gene promoter of tobaccoare induced also by specific compounds such as salicylic acid, and such,and the “rab 16” gene promoter is induced by the spraying of abcisicacid, a phytohormone.

In addition, for efficiently selecting cells transformed by introductionof a DNA of the present invention, the aforementioned recombinant vectorpreferably comprises a suitable screening marker gene, or is introducedinto the cells together with a plasmid vector comprising a screeningmarker gene. Examples of screening marker genes used for this purposeinclude hygromycin phosphotransferase gene, which is resistant to theantibiotic hygromycin; neomycin phosphotransferase gene, which isresistant to kanamycin or gentamicin; and acetyl transferase gene, whichis resistant to the herbicide, phosphinothricin.

In addition, the present invention provides transgenic plant cells intowhich a vector of the present invention has been introduced. There areno particular limitations on the cells into which a vector of thepresent invention is introduced, examples of which include the cells ofrice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane,rapeseed, soybean, sunflower, cotton, orange, grape, peach, pear, apple,tomato, Chinese cabbage, cabbage, radish, carrot, squash, cucumber,melon, parsley, orchid, chrysanthemum, lily, and saffron; however, treessuch as Eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, andyew are preferable. In addition, plant cells of the present inventioncomprise cultured cells, as well as cells present in a plant. Inaddition, protoplasts, shoot primordia, multiple shoots, and hairy rootsare also included.

Various techniques can be used to introduce an aforementioned expressionvector into host plant cells. Examples of these techniques includetransformation of plant cells by T-DNA using Agrobacterium tumefaciensor Agrobacterium rhizogenes for the transformation factor, directintroduction into a protoplast (by a method such as electroporation inwhich a DNA is introduced into plant cells by treating protoplasts withan electric pulse, fusion of protoplasts with liposomes and so forth,microinjection, and the use of polyethylene glycol), and the use of aparticle gun.

In addition, a desired gene can be introduced into a plant, by using aplant virus as vector. An example of a plant virus that can be used iscauliflower mosaic virus. Namely, after first preparing a recombinant byinserting the virus genome into a vector derived from E. coli and soforth, the desired gene is inserted into the virus genome. Such desiredgenes can then be introduced into a plant by cutting out the virusgenome modified in this manner from the recombinant with a restrictionenzyme, and inoculating into the plant (Hohn, et al. (1982), MolecularBiology of Plant Tumors (Academic Press, New York), p. 549, U.S. Pat.No. 4,407,956). The technique for introducing a vector into plant cellsor a plant is not limited to these, and includes other possibilities aswell.

There are no limitations on the required vector in the case of directinsertion into a protoplast. For example, a simple plasmid such as a pUCderivative can be used. Other DNA sequences may be required depending onthe method used to introduce the desired gene into plant cells. Forexample, in the case of using a Ti or Ri plasmid to transform plantcells, at least the sequence on the right end, and typically thesequences on both ends, of the T-DNA region of Ti and Ri plasmids mustbe connected so as to become an adjacent region of the gene to beintroduced.

When using an Agrobacterium species for transformation, a gene to beintroduced needs to be cloned into a special plasmid, namely anintermediate vector or a binary vector. Intermediate vectors are notreplicated in Agrobacterium species. Intermediate vectors aretransferred into Agrobacterium species by helper plasmids orelectroporation. Since intermediate vectors have a region that ishomologous with the T-DNA sequence, they are incorporated within the Tior Ri plasmid of Agrobacterium species by homologous recombination. Itis necessary for the Agrobacterium species used for the host to comprisea vir region. Normally, Ti or Ri plasmids comprise a vir region, and dueto its function, T-DNA can be transferred into plant cells.

On the other hand, since a binary vector can be replicated andmaintained in Agrobacterium species, when a vector is incorporated intoAgrobacterium species by a helper plasmid or electroporation, the T-DNAof the binary vector can be transferred into plant cells due to theaction of the vir region of the host.

Furthermore, intermediate vectors or binary vectors obtained in thismanner, as well as microorganisms such as E. coli and Agrobacteriumspecies that comprise them are also included in the present invention.

In addition, the present invention provides transgenic plants that havebeen redifferentiated from the aforementioned transgenic plant cells,transgenic plants that are progenies or clones of the transgenic plants,and breeding material of the transgenic plants. Such is a usefultransgenic plant in which cell wall components and cell morphogenesishave been altered. There are no particular limitations on the alterationof cell wall components in the present invention, and include variousquantitative and qualitative changes to create plants high in cellulose,low in lignin, having thick cell walls, thin cell walls, long and shortfiber lengths, etc. In addition, examples of cell morphology alterationsinclude, but are not limited to, changes in cell elongation and cellsize (quantitative changes in volume).

A transgenic plant of the present invention is useful as a plant havinga novel value such as increased plant growth as a result of increasingcell wall biosynthesis, altered fiber cell morphology, or increasedamounts of useful components in agricultural crops. In addition, it isalso useful as a plant having a novel value in developing new materialsby controlling cell wall biosynthesis, increasing the digestion andabsorption efficiencies of feed crops, changing fiber cell morphology,etc.

In the present invention, a “transgenic plant” refers to a plant havingthe aforementioned transgenic plant cells, and includes, for example, atransgenic plant regenerated from the aforementioned transgenic cells.Although the methods used to regenerate individual plants fromtransformed plant cells vary according to the type of plant cell, anexample of a method used in rice plants is the method of Fujimura et al.(Fujimura et al., Plant Tissue Culture Lett., 2, 74, 1995), the methodof Shillito et al. (Shillito et al., Bio/Technology, 7, 581, 1989) incorn plants, the method of Visser et al. (Visser et al., Theor. Appl.Genet., 78, 589, 1989) in potato plants, the method of Akama et al.(Akama et al., Plant Cell Rep., 12, 7, 1992) in Arabidopsis thaliana,and the method of Doi et al. (Japanese Patent Application No. Hei11-127025) in Eucalyptus plants. Transgenic plants produced according tothese methods or transgenic plants obtained from their breedingmaterials (such as seeds, tubers, or cuttings) are included in thepresent invention.

The present invention includes a process of producing a plant from aplant seed by introducing into a host a gene specifically expressed by aplant (particularly a tree) during cell wall formation and/orspecifically expressed during cellulose biosynthesis, a homolog thereof,or an expression vector comprising a promoter region that is contiguouswith these genes to obtain transgenic cells, regenerating a transgenicplant from said transgenic cells, and obtaining a plant seed from theresulting transgenic plant.

A process of obtaining a plant seed from a transgenic plant refers to aprocess in which, for example, a transgenic plant is acquired from arooting medium, replanted in a pot containing moist soil, and grown at aconstant temperature to form flowers, and finally seeds. In addition, aprocess of producing a plant from a seed refers to a process in which,for example, once a seed formed in a transgenic plant has matured, theseed is isolated, sowed on moist soil, and then grown at a constanttemperature and luminosity, to produce a plant.

The exogenously introduced DNA or nucleic acid in a transformed plantcan be confirmed by known methods, such as PCR or Southernhybridization, or by analyzing the nucleotide sequence of the plant'snucleic acid. To extract DNA or nucleic acid from a transformed plant,the known method of J. Sambrook et al. may be used (Molecular Cloning,2^(nd) edition, Cold Spring Harbor laboratory Press, 1989).

To conduct PCR analysis of a DNA of the present invention that exists ina plant, an amplification reaction is carried out using, as a template,nucleic acid extracted from the regenerated plant by the above-mentionedmethod. Amplification reaction may be carried out in a reaction mixturecontaining, as primers, synthesized oligonucleotides comprisingnucleotide sequences appropriately selected according to the nucleotidesequence of a DNA of the present invention. An amplified DNA fragmentcomprising a DNA sequence of the present invention may be obtained byrepeating several dozen cycles of the denaturation, annealing, andextension steps of the DNA amplification reaction. The respectiveamplified DNA fragments can be separated by, for example,electrophoresing the reaction solution containing the amplified productson agarose gel. DNA fragments corresponding to a DNA of the presentinvention can then be confirmed.

Having obtained a transformed plant in which a DNA of the presentinvention has been inserted into the chromosomes, one can obtain theplant's offspring by sexual or non-sexual reproduction. Also, it ispossible to mass-produce such plants by obtaining reproductive materials(such as seeds, fruits, cuttings, stem tubers, root tubers, shoots,calluses, and protoplasts) from the above plant, or its offspring orclones.

A stable supply of biomass, mainly cellulose, can be provided bycultivating a transgenic plant of the present invention on a largerscale using clone planting. At present, fossil resources are used inlarge amounts in industrial productions as raw materials and fuel(energy). With respect to alternative energy in particular, although thedirect combustion of wood biomass (for fuel) is routinely carried out indeveloping countries, a more effective approach would be possible byconverting the biomass into a more user-friendly form (such as alcohol,and specifically ethyl alcohol). In reality, ethanol is produced inBrazil and other countries, by alcohol fermentation of waste syrupobtained from squeezed sugar cane, and is used as an automobile fuel. Inthe U.S. and EU as well, there are a growing number of examples ofalcohol fermentation after initially hydrolyzing starch from sweetpotatoes and corn into glucose. In August 1999, the U.S. announced that,“the rate of biomass energy utilization will be increased to 10% of allprimary energy by the year 2010”. One of the objectives is to usegasoline mixed with ethanol refined from biomass. A specific example is“gasohol” (a 10% blend of ethanol in gasoline) made from corn. Gasoholis used in 20 states, mainly by those in the corn belt, and currentlyaccounts for about 1% of all automobile fuel used in the U.S. Gasoholaccounts for 40% of the gasoline share in certain states where sugarcane is cultivated. All U.S. automobile manufacturers have certified theuse of gasohol as fuel, and more specifically, General MotorsCorporation and DaimlerChrysler Corporationrecommend its use. In the EU,a project is underway to increase the share of recyclable energy to 12%of all energy, with the aim of reducing levels of greenhouse gases to 8%of the level of 1990 by the year 2010. This project has set the goal ofsubstituting biofuel (fuel derived from biological resources) for 5% ofall fossil fuels by 2005, as an alternative automobile fuel. The EU'senergy utilization plan calls for the use of solar cells (degree ofcontribution: 1%), wind power (19%), and biomass-cogeneration (80%),thus indicating the considerable expectations being placed on biomass.In addition, cultivation of biocrops for energy utilization is expectedto account for the largest land utilization area by 2015. On the otherhand, from the viewpoint of increasing food production, large-scaleconsumption of grains and potatoes as industrial raw materials would belimited in the future. Thus, if it were possible, for example, to growlarge numbers of the present invention's Eucalyptus trees having a highcellulose content, it would be possible to obtain glucose by hydrolysisor enzyme degradation (cellulase) using the resulting lignocellulose asraw material, and in turn enable large-scale production of ethanol byalcohol fermentation. Basic technology for such processes has alreadybeen established. Moreover, the technology for producing biodegradableplastics (polylactic acid) using glucose as raw material is alreadyestablished, and practical applications using potato starch isprogressing on an of industrial production scale. In the future however,it is predicted that biomass from trees will become the mainstreamreplacing grains, which is a food item. Furthermore, although it will benecessary to overcome technical problems in the future to achievepractical application of lignin, applications in plastics and adhesivesare expected. In addition, from the viewpoint of energy, although ligninis contained in waste liquid (referred to as black liquor) following itschemical decomposition in the production of pulp in the paper industry,it is being used as factory fuel after extracting the required chemicalsfrom the waste liquid. In other words, a portion of fuel is alreadydependent on wood biomass.

In addition to conventional use as raw materials, there is also aconsiderable potential for creating an alternative energy to petroleumthrough biomass conversion, as well as the development of new plasticsfrom cellulose and hemicellulose (both being technically possible), as aresult of stable and large-scale cultivation of wood biomass and therecycling of that wood biomass through afforestation as in the presentinvention. Moreover, the spread of wood biomass will contribute tosolving energy security problems and environmental issues, whilesimultaneously leading to the development of new industries, includingagricultural forestry, and the creation of employment opportunities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression intensities of major gene clusters inEucalyptus reaction wood. Two types of mRNA extracted from Eucalyptusreaction wood and normal wood were each labeled with two types offluorochromes (cy3, cy5); hybridization was carried out using thesemRNAs as probes in an oligo microarray analysis. The images resultingfrom scanning fluorescent intensity were analyzed with analyticalsoftware (Luminator Ver. 1.0, Rosetta). All repeated experiments wereintegrated to a statistical reliability of 99.9%, and the relativeexpression intensities of the major gene clusters in Eucalyptus reactionwood were graphed. In the photo, +(red) indicates genes for which asignificant increase in expression was observed, +(green) indicatesgenes for which a significant decrease in expression was observed,while+(blue) indicates genes for which changes in expression were notobserved.

BEST MODE FOR CARRYING OUT THE INVENTION

Although the following examples provide a more detailed explanation ofthe present invention, the present invention is not limited thereto. Theexperimental procedures were carried out in accordance with “Cloning andSequencing” (Watanabe, I., Sugiura, M., ed., Norin-Bunka Publishing(1989)) and “Molecular Cloning” (Sambrook, et al. (1989), Cold SpringHarbor Laboratory Press) unless indicated otherwise.

EXAMPLE 1 Production of a Eucalyptus EST Database

(1) Extraction of RNA from Eucalyptus

The thickly grown part of the trunk (secondary wall hypertrophic band;tissue rich in cambium), leaves, and roots, were selected as Eucalyptustissue for extraction, envisioning gene expression by variouscircumstances such as tension stress and stress due to exposure to saltsolutions. The method described in Hiono, et al. (Japanese PatentApplication No. Hei 6-219187) was used as the basic extractionprocedure. As an example of this method, the following provides adetailed explanation of the RNA extraction method using Eucalyptus rootobtained by a hydroponic cultivation.

Young Eucalyptus (Eucalyptus camaldulensis) plants grown for two monthswere transferred to a hydroponics tank. Hydroponic cultivation wascarried out using the culture medium of Hoagland-Amon, et al. Thecomposition of the hydroponic culture medium was: 5.0 mM KNO₃, 3.0 mMCa(NO₃)₂, 2.0 mM NH₄H₂PO₄, 2.0 mM MgSO₄, 47 μM H₃BO₃, 9.0 μM MnCl₂, 36μM FeSO₄, 3.1 μM ZnSO₄, 0.16 μM CuSO₄ and 75 μM (NH₄)₆Mo₇O₂₄. Thismedium was prepared using desalinated water, and the pH was adjusted to6.0 daily with 0.1 M NaOH or KOH. Moreover, the whole culture medium wasreplaced once a week. When conducting stress treatment, a culture mediumto which NaCl was sequentially added to a final concentration of 50,100, 200, and 300 mM from day 1 to day 4 was used for the stresstreatment group, while a culture medium to which NaCl was not added wasused for the control group. Ten grams of the root were cut into smallpieces and homogenized in liquid nitrogen on day 4. This was thentransferred to a 50 ml centrifuge tube (NUNC), and homogenized for 5minutes with a homogenizer after adding 10 g of glass beads. Solventextraction of the homogenized sample was repeated (about three times),until the supernatant was colorless using a methanol solution comprisingdithiothreitol (1 mg/ml). Following completion of extraction, the samplewas freeze-dried. The freeze-dried sample was mixed with 25 ml of pH 9100 mM CHES buffer (to which 20 mg of dithiothreitol and 10 mM vanadylribonucleoside compound solution were added immediately prior to use)and incubated for 30 minutes at 65° C. After incubation, 5 M aqueoussodium chloride solution and 10% CTAB solution were added to the samplesolution, so as to make the sodium chloride concentration 1.4 M, and theCTAB concentration 1% (w/v). After mixing the sample solution well andincubating for 10 minutes at 65° C., an equal volume of chloroform:isoamyl alcohol (24:1) solution was added and this was gently butthoroughly mixed. After mixing, the supernatant was recovered bycentrifugation. 55% by volume of isopropanol was added to thesupernatant followed by cooling with ice for 1 hour. A precipitate wasobtained by centrifugation, and phenol extraction was carried out afterdissolving the precipitate in water. 10% by volume of 3 M sodium acetateand 60% by volume of isopropanol were added to the supernatant followingphenol extraction, and after mixing well, the precipitate was recoveredby centrifugation. After dissolving the precipitate in sterile water, 12M lithium chloride solution was added to a final concentration of 3 M,and after mixing well, the solution was cooled with ice for 1 hour. AnRNA precipitate was recovered by centrifugation, and after washing anddrying, the RNA was finally dissolved in 100 μL of water to obtain atotal RNA fraction. As a result, 610 μg each of total RNA were obtainedfrom the roots of the stress treatment group and control group. mRNA waspurified from the total RNA fraction using the PolyATract mRNA IsolationSystem III & IV Kits (Cat. Nos. Z5300 and Z5310, Promega, USA). As aresult, from the 610 μg of total RNAs, 1.3 μg of mRNA were obtained fromthe sample of the stress treatment group and 1.8 μg of mRNA wereobtained from the control sample.

(2) Construction of cDNA Library

cDNA was synthesized using the Smart cDNA Library Construction Kit(Clontech) from Eucalyptus mRNA derived from each of the tissues andcircumstances according to the method described in (1) above, toultimately construct a phagemid library. The genomic DNA libraryproduced in this manner comprised independent clones of 1×10⁶ pfu ormore each. Furthermore, library amplification was carried out on aportion of the constructed library. Clone analysis was done withoutamplifying.

(3) Deciphering cDNA Clones and Database Construction

Clones were randomly selected from the Eucalyptus phagemid cDNA libraryderived from each tissue, and after purifying the plasmids, an enzymereaction was carried out using the Dye Terminator Sequence Kit(Amersham) followed by acquisition of nucleotide sequence data using alarge-scale, high-speed sequencer (Amersham).

The data was analyzed using analytical software after deleting knownplasmid sequences, and homologous sequences were extracted by aclustering procedure. Subsequently, a comparative search was done usingthe entire database of GenBank, U.S.A, one of the genetic informationdatabases, to roughly predict (annotation) the function.

The size of the final database is shown in Table 1. TABLE 1 Total numberof nucleotide Number of Total Subject sequences clusters (total numberof No. tissue deciphered constituent no.) singlets OJI001, Trunk 206452762 (16026) 4619 OJI005 OJ1004 Leaf 10171 1021 (7574) 2597 OJI002, Root10726 1511 (7169) 3557 OJI003 OJI001-005 All tissue 41542 4660 (34206)7336

EXAMPLE 2 Extraction of Genes Specifically Expressed in EucalyptusReaction Wood Tissue

(1) Production of a Eucalyptus Trunk-Specific Oligo Microarray

A Eucalyptus oligo microarray was produced targeting the entire sequenceexcluding the overlapping sequences from 0JI001 and 0JI005 according tothe Eucalyptus EST database shown in Table 1. Actual production of themicroarray was commissioned to Agilent Technologies, Inc. (Japaneserepresentative: Yokogawa Analytical Systems Inc.). Details are describedin the following web site:http://www.agilent.com/cag/country/JP/products/PCol494.html.

The Eucalyptus oligo microarray produced in this manner comprised 8400oligo DNA, and was able to cover a majority of the genes recognized tobe expressed in the Eucalyptus trunk.

(2) Extraction of Genes Specifically Expressed in Eucalyptus ReactionWood Forming Tissue by Microarray Analysis

The biosynthesis of cellulose (a major component of the cell wall) inbroad-leaved trees in particular, is known to involve the formation oftissue whose cell wall cellulose content roughly doubles as a result ofthe external tension stress. Comprehensive determination of the seriesof genes responsible for cellulose biosynthesis in particular, duringcell wall formation, is possible by analyzing this tissue using theaforementioned genomic analysis. In addition, differences in expressionof each component gene can also be determined by combining geneexpression analysis by microarray analysis using EST data.

Tension wood has long been known as a characteristic phenomenon thatresults from the aforementioned external mechanical stress. Reactionwood of broad-leaved trees refers to the special secondary wood that isformed as a result of a tree trunk having detected a change in thedirection of gravity, when responding to an external tension stress.Compared with ordinary wood, a cellulose increase and lignin andhemicellulose decrease is seen in cell wall components. In addition,morphological observations reveal that xylem distribution densitydecrease to half of that of ordinary wood, and that the leaning angle ofmicrofibrils in the cell walls became nearly parallel with the axialdirection of cell growth. Moreover, fiber length is observed to increaseby roughly 20%. Although the details are unknown, it is thought that agrowth strain results, by which the trunk, at locations wherelongitudinal growth is already over, attempts to return to the correctposition in response to the leaning. As the leaning persists, a singlebranch finally begins new vertical elongation, in place of the maintrunk. However, during the time the trunk attempts to rise upward inresponse to the leaning, tissue is formed in which cellulose contentbecomes extremely high in the tissue at the top of the leaned trunk.When a cross-section of the trunk is observed, semi-transparent tissuethat is different from ordinary wood tissue can also be visuallyobserved easily. Although general descriptions on reaction wood arealways disclosed in technical literature relating to wood, a paper byBaba, et al. (Mokuzai Gakkaishi (Academic Journal of Wood and Lumber)42, 795-798, 1996) has detailed data on the chemical and histologicalproperties of reaction wood as related to Eucalyptus.

The present inventor extracted total RNA from a cloned line ofEucalyptus camaldulensis (CPT1), in accordance with the RNA extractionmethod described in Example 1, using normal wood and reaction wood. Aspecific reaction wood tissue is formed in the upper portion of thetrunk, by artificially pulling and tilting an ordinary growing trunk toan angle of about 45 degrees. mRNA was purified from the total RNAobtained from ordinary wood and reaction wood, using the PolyATract mRNAIsolation System (Promega). The two types of mRNA obtained in thismanner were then each labeled with two types of fluorochromes (cy3, cy5)followed by hybridization using them as probes in oligo microarrayanalysis (FIG. 1). The hybridization method, including labeling, wasperformed in accordance with the analysis protocol as directed byAgilent Technologies, Inc.

As a result, genes were broadly classified into a gene cluster with apredominantly high expression, a gene cluster with a low expression, anda gene cluster with a virtually unchanged expression, in Eucalyptusreaction wood as compared to ordinary trunks. In particular, the genecluster that demonstrates a predominantly high expression, is thought tobe involved in cell wall component biosynthesis and wood fiber cellmorphogenesis. More specifically, this gene cluster is thought to bedirectly involved in the expression of traits such as high cellulosecontent and low lignin content that are characteristic to reaction wood.

INDUSTRIAL APPLICABILITY

Use of DNA of the present invention enabled the artificial control ofwood biomass production in trees. Particularly it was possible to changethe quantity and quality of the essence of wood mass-the cell wallcomponents (cellulose, hemicellulose, and lignin). Furthermore, fibermorphology (wood fiber cell elongation) could be freely altered. Namely,quantitative increases and qualitative modifications of essential woodbiomass, and the resultant expansion of uses in future energies and inapplications as industrial raw materials are expected, hopefullyreplacing the current fossil materials in various fields.

1. A DNA whose expression increases during plant cell wall biosynthesisand wood fiber cell morphogenesis, wherein the DNA is described in (a)or (b) below: (a) a DNA that hybridizes under stringent conditions witha DNA comprising a nucleotide sequence described in any one of SEQ IDNOs: 1 to 862; or, (b) a DNA encoding a protein having 50% or morehomology with a protein comprising an amino acid sequence encoded by theDNA of (a).
 2. The DNA of claim 1, wherein expression increases in plantreaction wood forming tissue.
 3. A DNA whose expression decreases duringplant cell wall biosynthesis and wood fiber cell morphogenesis, whereinthe DNA is described in (a) or (b) below: (a) a DNA that hybridizesunder stringent conditions with a DNA comprising a nucleotide sequencedescribed in any one of SEQ ID NOs: 863 to 1731; or, (b) a DNA encodinga protein having 50% or more homology with a protein comprising an aminoacid sequence encoded by the DNA of (a).
 4. The DNA of claim 3, whereinexpression decreases in plant reaction wood forming tissue.
 5. The DNAof claim 1 or 3, wherein the plant is Eucalyptus.
 6. A DNA encoding aprotein comprising an amino acid sequence in which one or more aminoacids are substituted, deleted, added and/or inserted in an amino acidsequence encoded by the DNA of claim 1 or
 3. 7. A promoter DNA of theDNA of claim 1 or
 3. 8. A DNA described in any one of (a) to (e) below:(a) a DNA encoding an antisense RNA complementary to a transcriptionproduct of the DNA of claim 1 or 3; (b) a DNA encoding an RNA havingribozyme activity that specifically cleaves a transcription product ofthe DNA of claim 1 or 3; (c) a DNA encoding an RNA that suppressesexpression of the DNA of claim 1 or 3 by RNAi effects; (d) a DNAencoding an RNA that suppresses expression of the DNA of claim 1 or 3 byco-suppression effects; and, (e) a DNA encoding a protein having adominant negative trait against a transcription product of the DNA ofclaim 1 or
 3. 9. A recombinant vector comprising the DNA of claim 1 or3.
 10. A microorganism retaining a plasmid comprising the vector ofclaim
 9. 11. A transgenic plant cell introduced with the vector of claim9.
 12. A transgenic plant that is re-differentiated from the transgenicplant cell of claim
 11. 13. A transgenic plant that is a progeny or aclone of the transgenic plant of claim
 12. 14. A breeding material ofthe transgenic plant of claim
 12. 15. A breeding material of thetransgenic plant of claim
 13. 16. A recombinant vector comprising theDNA of claim
 8. 17. A microorganism retaining a plasmid comprising thevector of claim
 16. 18. A transgenic plant cell introduced with thevector of claim
 16. 19. A transgenic plant that is re-differentiatedfrom the transgenic plant cell of claim
 18. 20. A transgenic plant thatis a progeny or a clone of the transgenic plant of claim
 19. 21. Abreeding material of the transgenic plant of claim
 19. 22. A breedingmaterial of the transgenic plant of claim
 20. 23. A microorganismretaining a plasmid comprising the promoter DNA of claim 7.